Our long term objective is to investigate the molecular mechanisms that control the proliferation and differentiation of human neural crest stem cells (hNCSCs) and use these cells for regenerative medicine. Embryonic NCSCs are pluripotent and NCSCs have been shown to persist in the adult in at least two differentiated tissues, the enteric nervous system of the gut and the skin and hair follicles. Hair follicles are a niche for NCSCs in the skin. Bone morphogenic proteins (BMPs) play a critical role in embryonic NCSC self-renewal and differentiation. NCSCs have been well characterized in chick and murine systems. Despite the importance of neural crest cell biology in development and disease, the isolation of NCSCs of human origin has been elusive. We hypothesize that human hair follicle NCSCs retain pluripotency and have similar functions as embryonic NCSCs. We have recently identified a stem cell population in human hair follicles. These cells are distinctively different from epithelial or melanocyte stem cells. They are clonogenic, capable of self-renewal and pluripotent. They express NCSC markers and BMP-4 as well as BMP receptors but not lineage specific markers. These cells are located in the bulge region of human hair follicles. We propose to characterize the bulge hNCSCs in vitro and in vivo. Aim 1: Characterize hNCSCs in human hair follicles. We will further characterize the pluripotent of these bulge hNCSCs, define their genetic signature and identify surface markers that can be used to directly isolate these cells from fresh tissues. We will study the role of BMP signal pathway in the proliferation and differentiation of bulge hNCSCs. Aim 2: Study the physiological function of hNCSCs in vivo. We will test the hypothesis that bulge hNCSCs have a similar biological function as embryonic NCSCs. We will study whether bulge hNCSCs follow microenvironmental neural crest cues to develop into appropriate cell types and their physiological functions in the skin and hair follicles in vivo. Aim 3: Determine the signals for hNCSCs to differentiate to melanocytes. We will study the mechanism underlying lineage determination for hNCSCs, particularly staged differentiation of hNCSCs to melanoblasts to mature melanocytes. We will study the interaction of Wnt3a, stem cell factor and endothelin-3 with Mitf during this process. We expect that bulge hNCSCs retain the pluripotency and function of embryonic NCSCs. They contribute to normal human skin and hair follicle homeostasis. The proposed studies will establish a novel model to study human NCSCs in the somatic tissues. This model can be used to study various signalling pathways that are essential for hNCSC self-renewal and lineage determination, the physiological functions of hNCSCs in adult tissue, and the potential use of these cells for regenerative therapy. Also, because many diseases, including some cancers, are known to involve neural crest cells, the models developed in these studies may be useful in testing the etiologies of these diseases. PUBLIC HEALTH RELEVANCE. Stem cell research holds tremendous promise for regenerative medicine. The goal of this application is to reveal the molecular mechanisms underlying the proliferation and differentiation of human neural crest stem cells in the hair follicles. The proposed studies may lead to potential use of these cells in regenerative therapy and establish a novel model to study the etiology of neural crest cells related human diseases.